Method for the production of pure virally inactivated butyrylcholinesterase

ABSTRACT

The present invention provides a method for purifying butyrylcholinesterase from various biological fluids. Biological fluids include, e.g., blood, blood fractions, plasma, and bioreactor broths, and other such mixtures containing butyrylcholinesterase. In one embodiment, the invention provides a method for the production of purified, virally inactivated butyrylcholinesterase by contacting a biological fluid containing butyrylcholinesterase with a cationic exchange chromatography material, with an affinity chromatography material, and treating the fluid with solvent detergent. The resulting purified butyrylcholinesterase can also be subjected to a pasteurization step, and formulated in a sodium chloride/sodium phosphate solution for storage or lyophilization.

This application is a divisional application of Ser. No. 10/10/061,233,filed Feb. 4, 2002, the entire content of which is hereby incorporatedby reference.

FIELD OF THE INVENTION

The present invention provides a method for the isolation andpurification of butyrylcholinesterase from complex biological fluidssuch as plasma. More specifically, the present invention provides amethod for purifying and disinfecting butyrylcholinesterase frombutyrylcholinesterase-containing biological fluids, e.g., Cohn FractionIV-4 or IV-1, comprising subjecting such fluids to cationic exchangechromatography, affinity chromatography, a solvent detergent treatment,and pasteurization. As discussed more fully below, the order of thosesteps can be varied.

BACKGROUND OF THE INVENTION

Butyrylcholinesterase is an enzyme found mainly in plasma. Although thenormal physiological role of butyrylcholinesterase is unknown,butyrylcholinesterase has been shown to metabolize acetylcholine,degrade cocaine, and inactivate anaesthetic drugs and muscle relaxants,including succinylcholine, succinylcholine-like compounds and mivacurium(Gorelick et al., Drug Alcohol Depend., 48(3): 159-65, 1997; Stewart etal., Clin. Pharmacol. Ther., 25: 464-8, 1979; Krasowski et al., Can. J.Anaesth., 44:525-34, 1997; Jatlow et al., Anesth. Anag., 58:235-8,1979). Butyrylcholinesterase has also been shown to act as an antidoteto nerve gas and organo-phosphorus compounds (Ashani et al., Biochem.Pharmacol., 41:37-41, 1991; Broomfield et al., JPET, 259:633-8, 1991;Doctor et al., New Approaches to Medical Protection Against ChemicalWarfare Nerve Agents In Chemical Warfare Agents: Toxicity at Low Levels,pp. 191-214, Lewis Publishers, Inc., 2001; Ashani et al., DrugDevelopment Research, 50:298-308, 2000).

Previous methods for isolating butyrylcholinesterase involved eitherammonium sulfate precipitations or electrophoresis, which resulted inyields of butyrylcholinesterase of about 10% and purities of 10% or less(Goedde et al., Humangenetik., 1:311-8 1965; Haupt et al., Blut.,14(2):65-75, 1966). Present methods employing chromatography of plasmaresult in yields of butyrylcholinesterase ranging from 30-40% to 63%(Grunwald et al., J. Biochem. Biophys. Methods, 34(2):123-35, 1997;Lockridge et al., J. Biol. Chem., 287:12012-8, 1982). In order toevaluate butyrylcholinesterase for its therapeutic and pharmacologicalproperties, large quantities of purified, virally inactivatedbutyrylcholinesterase are needed.

Within the art, there remains a need for a method whereby largequantities of virally inactivated butyrylcholinesterase can be isolatedfrom biological fluids such as plasma in a highly purified form. Untilnow, there have been no commercially viable, easily performed methods toefficiently and economically produce large quantities of purified,virally inactivated butyrylcholinesterase from biological fluids. Themethods of the present invention address that need.

SUMMARY OF THE INVENTION

To satisfy the need in the art, we have developed a versatile,commercially viable method for producing purified, virally inactivatedbutyrylcholinesterase at high yields and purity. Specifically, thepresent invention provides a novel method for recovering purified,virally inactivated butyrylcholinesterase from a biological fluid suchas plasma, Cohn plasma Fraction IV-4 or IV-1, analogous blood fractions,or fluids from bioreactors. The various embodiments of the methodcomprise subjecting a butyrylcholinesterase-containing fluid to a seriesof steps including cationic exchange chromatography, affinitychromatography, solvent detergent treatment, and pasteurization. Thosesteps need not be performed in that order, and the methods can besuccessfully performed with duplication of one or more of those steps.

DETAILED DESCRIPTION OF THE INVENTION

The inefficient and complex aspects of butyrylcholinesterasepurification and viral inactivation can be eliminated using a simpletechnique employing one or more steps incorporating cation exchangechromatography and affinity chromatography. The method of the inventioncan be used to produce purified and virally inactivatedbutyrylcholinesterase from plasma or plasma fractions in yields rangingfrom about 70% to about 80%. The term pure or purified is used herein torefer to a purity of greater than about 70%. Preferred embodimentsafford butyrylcholinesterase in purity of about 80% or greater. The morepreferred embodiments afford butyrylcholinesterase in purity of about90% or greater.

The purity of butyrylcholinesterase is determined by SDS-PAGE analysisand by the specific activity of the final product. Enzymatic activity ofthe purified butyrylcholinesterase is expressed as mg/mg or units/mg.

Butyrylcholinesterase can be isolated frombutyrylcholinesterase-containing sources such as commercially availableplasma, plasma fractions such as Cohn Fraction IV-1 or Cohn FractionIV-4, a mixed plasma fraction of Cohn Fraction IV-1 and IV-4,recombinant sources, and other biological samples. If plasma is used,the plasma can be treated to produce Cohn Fraction IV-4 or IV-1, orplasma fractions of similar composition, as set forth in Cohn et al. (J.Amer. Chem. Soc., 68:459, 1946), or by other methods known in the art.

In accordance with one embodiment of the invention, there is provided amethod for producing purified, virally inactivated butyrylcholinesterasefrom human plasma, in particular Cohn plasma Fraction IV-4. Onepreferred example of the method comprises subjecting a solution of Cohnplasma Fraction IV-4 to cation exchange chromatography followed byaffinity chromatography. A solvent detergent treatment step may be addedto inactivate lipid-enveloped viruses; and one or more of thechromatographic steps can be duplicated. The purification portion of themethod is versatile, and the order of performing the various steps isnot crucial. The purified butyrylcholinesterase is then pasteurized as ageneral pathogen inactivation step and to inactivate viruses.

The cation exchange chromatography step is performed using any one of awide variety of cation exchange materials, for example cation exchangerslinked to supports such as agarose, dextran, cellulose, polyacrylamide,polystyrene, acrylic polymers, vinyl polymers, and silica. Cationexchangers such as carboxymethyl and sulfopropyl moieties can be linkedto, e.g., agarose to produce carboxymethyl-agarose (e.g., CM-SEPHAROSE7)and sulfopropyl-agarose (e.g., SP-SEPHAROSE7), respectively.CM-SEPHAROSE7 is a preferred cation exchange material in methods of thepresent invention.

Cation exchange chromatography can be performed by any of the knownmethods in the art. Those skilled in the art will appreciate that anyconventional format for effectively exploiting cation exchangechromatography materials will be suitable. In a preferred embodiment,column chromatography is used. In such an embodiment, the cationexchange chromatography column is packed with CM-SEPHAROSE7 andequilibrated with buffer, preferably about 25 mM sodium acetate, to a pHranging from about 4.8 to about 5.2 and a conductivity ranging fromabout 0.85 to about 6.0 mS. Other equilibration buffers encompassed bythe method of the invention include but are not limited to sodiumcitrate and sodium phosphate.

The fall through from the cation ion exchange column contains thebutyrylcholinesterase. The resulting biological fluid is adjusted to pHof about 6.0 to about 8.5, more preferably to a pH of about 7.5. Thefall through is then concentrated about 5 to about 10 fold by methodsknown in the art, for example by ultrafiltration or diafiltration, andadjusted to a pH ranging from about 6.0 to about 8.5 and a conductivityranging from about 6.0 to about 15 mS.

At this stage of the butyrylcholinesterase purification procedure, thefall through concentrate can be solvent-detergent treated before orafter an affinity chromatography step. Solvent-detergent treatmentinvolves mixing the fall through or eluate with a solvent such asTri-n-Butyl Phosphate (TnBP) mixed with a detergent such as thenon-ionic detergent Tween-80 or Triton X-100, or combinations thereof,at a concentration ranging from about 0.3% to about 1.0% (w/v) for eachsolvent-detergent used. Solvent-detergent treatment of the eluate afteraffinity chromatography is preferable because the volume of eluate beingtreated is much smaller.

The fall through concentrate from the cation exchange chromatographystep is then subjected to affinity chromatography. Again, anyconventional method for performing a purification step using affinitychromatography material is acceptable; however, the preferred method iscolumn chromatography. Affinity chromatography materials include, butare not limited to, amino acid resins, peptide ligand resins, antibodyresins, carbohydrate resins, avidin/biotin resins, dye resins,glutathione resins, hydrophobic resins, immunochemical resins, lectinresins, nucleic acid resins and nucleotide/coenzyme resins. The term“peptide ligand resins” is used to refer to affinity chromatographymatrices to which peptides (i.e., chains of two or more amino acids) arecoupled.

In a preferred embodiment of the invention, affinity chromatography isperformed using any one of a wide variety of affinity chromatographyligands linked to conventional supports. Conventional supports arematrices such as gels or resins and include, but are not limited to,agarose, dextran, cellulose, polystyrene, acrylic resins, acrylamides,vinyl resins, and cross-linked and/or derivatized variations thereof.One of skill in the art will appreciate that the affinity chromatographysupport material can be chosen from a variety of commercially availablematerials, and will be selected from among the various materials suchthat it has the requisite pore size to accommodatebutyrylcholinesterase.

A preferred affinity chromatography material is that in which thesupport is agarose covalently binding procainamide. Another preferredaffinity chromatography material is an agarose support covalentlybinding a butyrylcholinesterase-binding peptide. Examples of suchpeptides are presented below.

Commercially available examples of suitable supports include SEPHAROSE7and SEPHADEX7 affinity chromatography resins. One of skill in the artwill appreciate that many of the supports that are commonly employed inaffinity chromatography materials, and which have an appropriate poresize for butyrylcholinesterase will be useful in the present invention.Those supports may be purchased, and preferred ligand can besubsequently coupled to the resin.

As stated above, various materials can be used to perform affinitypurification of butyrylcholinesterase from biological fluids. Forexample, procainamide affinity chromatography material can be employed.Another option is a chromatography material to which is bound anantibody that binds butyrylcholinesterase. Alternatively, other proteinsor compounds such as peptide ligands or inhibitors that bindbutyrylcholinesterase can be coupled to the resin. Methods for usingthose additional materials (i.e., antibodies, substrates, compounds,inhibitors, etc.) for affinity chromatography are known within the art.

In one embodiment, the present invention provides a method for producingpurified, virally inactivated butyrylcholinesterase comprising:contacting a butyrylcholinesterase-containing biological fluid with acation exchange chromatography material; solvent-detergent treating thebiological fluid; contacting the biological fluid with abutyrylcholinesterase-binding affinity chromatography material;recovering bound butyrylcholinesterase from the affinity chromatographymaterial; and pasteurizing the recovered butyrylcholinesterase. Althoughit will be appreciated that certain steps will be performedsequentially, it is an advantage of the present invention that severalof the foregoing steps can be interchanged in order without sacrificingthe effectiveness of the method. Moreover, the purity of the recoveredbutyrylcholinesterase can be improved by repeating one or more of thesteps, particularly the affinity chromatography step.

The method affords the added versatility that when duplicating suchsteps, different materials can be used. Thus, if one chooses to repeatthe affinity chromatography step, different chromatography media can beemployed in the two different steps.

Butyrylcholinesterase can be affinity purified frombutyrylcholinesterase containing sources using peptide ligands that bindbutyrylcholinesterase. In such an embodiment, peptide ligands that bindbutyrylcholinesterase are first identified. This can be done byscreening combinatorial peptide libraries using labeledbutyrylcholinesterase. Butyrylcholinesterase can be labeled using any ofthe conventional methods in the art, such as radiolabeling. Peptideligands that are positive for binding butyrylcholinesterase can bedetected by using known methods in the art. For example, a conventionalenzyme activity assay using butyrylcholine and DTNB(5,5-Dithiobis-2-Nitrobenzoic Acid) can be used. The peptide ligandsfound to bind butyrylcholinesterase can then be sequenced, andreproduced for isolating and purifying butyrylcholinesterase.

The positive peptide ligands can then be used in such an isolation andpurification process in otherwise conventional affinity chromatographymethods. In one embodiment, the ligands identified as having therequired affinity for butyrylcholinesterase are immobilized on anaffinity chromatography support or matrix material. Any common affinitychromatography support or matrix material with the requisite pore sizewill likely be suitable.

In a preferred embodiment, the ligand(s) are covalently attached to anaffinity chromatography matrix to create a matrix-ligand composite. Abiological fluid containing butyrylcholinesterase is contacted with thematrix-ligand composite, and butyrylcholinesterase is concentrated onthe composite. Extraneous matter and undesired components from thebiological fluid are washed away; and the butyrylcholinesterase issubsequently eluted from the composite with appropriate buffers andsolutions, and the butyrylcholinesterase is recovered.

The term “biological fluid” is used herein to refer to an aqueous fluidor mixture containing various biological constituents and contaminantsin combination with butyrylcholinesterase. In short, a biological fluid,as used herein, is an aqueous mixture of impure butyrylcholinesterase.The butyrylcholinesterase in the biological fluid is either natural orproduced from recombinant sources. Examples of biological fluids includeblood; blood fractions, plasma, plasma fractions, extracts, andisolates; cell or tissue homogenates, extracts, or isolates; andbioreactor broths or other reaction mixtures suitable for making and/orrecovering butyrylcholinesterase. Preferred biological fluids forrecovering natural butyrylcholinesterase are Cohn Fractions IV-4, CohnFraction IV-1, Precipitate IV (hereinafter “PPT. IV”; also referred toin the art as Precipitate B or PPT. B) from the Kistler-Nitschmannfractionation, and combinations thereof. For a description of PPT. IVsee, Kistler P. and Nitschmann H., Vox. Sanguinis, 7, 414 (1960).

As discussed below, the various biological fluids, such as plasmafractions, can be reconstituted in water or other appropriate aqueoussolvents to achieve the desired density, product concentration, and thelike. Selection of aqueous solvent and the amount used forreconstitution will vary depending upon the biological fluid employed,and can be routinely determined by one of ordinary skill in the art.

We have identified the following peptides as preferred peptide affinityligands for the concentration, separation, and purification ofbutyrylcholinesterase: AKDQIP (SEQ ID NO: 1) (alanine, lysine, asparticacid, glutamine, isoleucine, proline), AKGDQN (SEQ ID NO: 2) (alanine,lysine, glycine, aspartic acid, glutamine, asparagine), WKDAVQ (SEQ IDNO: 3) (tryptophan, lysine, aspartic acid, alanine, valine, glutamine),GFVGXA (SEQ ID NO: 4) (glycine, phenylalanine, valine, glycine, X,alanine, wherein X is 2-naphthylalanine), GFHGXI (SEQ ID NO: 5)(glycine, phenylalanine, histidine, glycine, X, isoleucine, wherein X is2-naphthylalanine), AFTNGE (SEQ ID NO: 6) (alanine, phenylalanine,threonine, asparagine, glycine, glutamic acid), AFTNQE (SEQ ID NO: 7)(alanine, phenylalanine, threonine, asparagine, glutamine, glutamicacid), GTNYHQ (SEQ ID NO: 8) (glycine, threonine, asparagine, tyrosine,histidine, glutamine), AEVDPG (SEQ ID NO: 9) (alanine, glutamic acid,valine, aspartic acid, proline, glycine).

In still another embodiment of the present invention,butyrylcholinesterase can be concentrated from a biological fluid in asimple separation of one or more steps. That is, the peptide ligandsidentified above can be used in a highly versatile and effectiveone-step method for concentrating and isolating butyrylcholinesterasefrom biological fluids. Thus, the present invention provides a methodfor concentrating butyrylcholinesterase comprising contacting abutyrylcholinesterase-containing biological fluid with a peptide ligandaffinity chromatography material wherein the peptide ligand is selectedfrom the group consisting of: a) alanine, lysine, aspartic acid,glutamine, isoleucine, and proline; b) alanine, lysine, glycine,aspartic acid, glutamine, and asparagine; c) tryptophan, lysine,aspartic acid, alanine, valine, and glutamine; d) glycine,phenylalanine, valine, glycine, 2-naphthylalanine, and alanine; e)glycine, phenylalanine, histidine, glycine, 2-naphthylalanine, andisoleucine; f) alanine, phenylalanine, threonine, asparagine, glycine,and glutamic acid; g) alanine, phenylalanine, threonine, asparagine,glutamine, and glutamic acid; h) glycine, threonine, asparagine,tyrosine, histidine, glutamine; and i) alanine, glutamic acid, valine,aspartic acid, proline, glycine; and recovering butyrylcholinesterasebound to said peptide ligand chromatography material. The contactingstep can be a standard column chromatography method; and the recoverycan be performed by eluting the butyrylcholinesterase from the columnusing known buffers, salt solutions, and solvents.

The simple one-step separation method can be combined with one or morepurification and/or disinfection steps. Thus, the peptide ligandaffinity separation step can be coupled with a cation exchange step, asolvent detergent treatment step, and/or a pasteurization step.

Further, one or more of the purification steps (i.e., affinity or cationexchange) can be duplicated in a variation of the method. In duplicatingone or more of those steps, different media can be employed. Forexample, in duplicating the affinity separation step, it is possible touse a different peptide ligand or a different class of ligand (e.g.,procainamide).

Butyrylcholinesterase can be affinity purified using a variety ofaffinity chromatography media. In another preferred embodiment,procainamide affinity reagent (p-amino-N-(2-diethylaminoethyl)benzamide)is covalently coupled to 6-aminohexanoic acid agarose using EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) as a coupling reagent.The affinity chromatography column with procainamide affinity reagent isequilibrated with buffer, preferably 20 mM sodium phosphate and 0.1 Msodium chloride, to a pH ranging from about 6.0 to about 8.5 and aconductivity ranging from about 8 to about 15 mS. Other equilibrationbuffers encompassed by the method of the invention include but are notlimited to Tris and glycine buffers.

As stated above, the eluate from the affinity chromatography column canbe solvent-detergent treated. If solvent-detergent treatment isperformed at this stage of butyrylcholinesterase isolation, then thissolvent-detergent treated eluate is subjected to a second round ofaffinity chromatography, and the butyrylcholinesterase is eluted fromthe column with sodium chloride ranging from about 0.3 to about 0.5 M.Solvent-detergent treatment is performed as described above.

The methods of the present invention can be used to inactivate lipidenveloped or non-lipid enveloped viruses. Therefore, the purifiedbutyrylcholinesterase is pasteurized for a time period ranging fromabout 8 to about 72 hours, preferably from about 8 to about 24 hours,without substantial loss of butyrylcholinesterase activity.

The present methods afford viral inactivation with retention of at leastabout 80% butyrylcholinesterase activity even after pasteurization forabout 72 hours. In one embodiment, pasteurization is performed at about+60° C. for about 8 to about 12 hours in solutions of L-lysine (about0.1 to about 1.0 M) and sodium citrate (about 0.5 to about 1.0 M);sucrose (about 0.3 to about 0.6 M) and sodium citrate (about 0.6 toabout 1.0 M); or sucrose (about 0.6 to about 1.0 M) and glycine (about0.3 to about 0.5 M). Preferably, pasteurization is performed in asolution of sucrose (about 0.3 to about 0.6 M) and sodium citrate (about0.6 to about 1.0 M).

Esterolytic activity of butyrylcholinesterase was determined in astandard esterolytic assay with the substrate butyrylthiocholine.Retention of esterolytic activity of butyrylcholinesterase for about 24hours at about +60° C. was achieved in formulations of about 0.3 toabout 0.6M sucrose in varying combinations with about 0.6 to about 1.0 Msodium citrate as well as in a formulation of about 0.1 M lysine andabout 1.0 M sodium citrate, as preferred pasteurization formulations.The following Examples are provided to further illustrate a fewembodiments of the present invention. The Examples are presented forillustration only, and do not reflect or suggest any limitation on thescope of the invention.

EXAMPLE I Purification and Viral Inactivation of Butyrylcholinesterase

Materials: Cohn Fraction IV-4 paste obtained by the Cohn cold ethanolfractionation process of pooled human plasma was used in this example.The cation exchange materials used (CM-SEPHAROSE7, FAST FLOW7 ORSP-SEPHAROSE7, FAST FLOW7) are commercially available resins.

The affinity resin was prepared by coupling procainamide covalently toECH-SEPHAROSE 4B7 (Pharmacia) using EDC as a coupling reagent.Procainamide was added in 5-fold molar excess per gram of swollen gelrelative to the resin ligands. EDC was then added to a finalconcentration of about 0.1 M. The coupling procedure was performed indistilled water, adjusted to a pH ranging from about 4.5 to about 5.5with HCl. The procainamide/EDC mixture was rotated gently for about 24hours at room temperature, and the resin was subsequently washed withseveral cycles of high and low pH solutions (0.1 M acetate buffer, 0.5 MNaCl pH 4.0; 0.1 M Tris-HCl buffer; 0.5 M NaCl, pH 8.0), followed bywashes with distilled water. The resulting resin was stored in about 20%ethanol.

In this example, the method of the invention was performed as follows:1.0 kg of Cohn IV-4 was suspended in about 8.0 L of distilled water(optionally containing 1.0 mM EDTA) to form a mixture and stirred forabout 2 hours or overnight at about 4EC. Particulate matter such asinsoluble proteins and Celite, an additive added to collect CohnFraction IV-4, was removed by centrifugation at about 4000 g andclarified through 5 m, 1.0 m, 0.5 m and 0.2 m filters. After adjustingthe pH to a range from about 4.9 to about 5.1 with acetic acid and theconductivity to between about 0.85 to about 1.3 mS with distilled water,the mixture was loaded on a cation exchange chromatography column packedwith approximately 0.35 L-0.5 L of CM-SEPHAROSE7, which had beenequilibrated with about 25 mM sodium acetate (pH 4.9; conductivity 1.8mS; temperature 22EC), at a linear velocity of about 60 cm/H and aresidence time ranging from about 10 to about 12 minutes (ifSP-SEPHAROSE7 resin is used, the pH is about 5.2 and the conductivity isabout 6.0 mS). The column was washed with about 0.5 L of 25 mM sodiumacetate buffer (pH 4.9), and the wash was added to the fall through tocollect the butyrylcholinesterase quantitatively. The specific advantageof this chromatographic step was that the butyrylcholinesterase remainedin the fall through while greater than 90% of the contaminating proteinswere bound to the cation exchange material.

The fall through, which contains the butyrylcholinesterase, was adjustedto a pH of about 7.5 with about 1.0 M NaOH and then concentrated 5 to 10fold by ultrafiltration using a membrane with a molecular weight cutoffof about 100,000. After concentrating the fall through, the conductivitywas adjusted to about 9.5 mS with about 4.0 M NaCl.

The concentrate from above was then loaded onto an affinitychromatography column packed with approximately 0.05-0.075 L ofprocainamide (PAM) affinity resin, which had been equilibrated withabout 20 mM sodium phosphate and about 0.1 M sodium chloride (pH 7.5;conductivity 10.5 mS), at a linear velocity of about 50 cm/H. The columnwas washed step-wise with about 0.1, 0.15, 0.175 and 0.2 M NaCl in about20 M sodium phosphate (pH 7.5) and then eluted with about 0.4 L of 0.5 MNaCl and 20 mM sodium phosphate (pH 7.5).

At this stage of the butyrylcholinesterase purification procedure, theeluted butyrylcholinesterase was treated with solvent-detergent.Solvent-detergent treatment involved mixing the PAM affinity eluate fallthrough with about 1% (v/v) Tween-80 and about 0.3% (v/v) TnBP.

The eluate was then subjected to a second round of PAM affinitychromatography and washed and eluted as described above. The yield andpurity of the isolated butyrylcholinesterase was calculated to be about80% and about 80-90%, respectively.

To inactivate both lipid and non-lipid enveloped viruses, such asporcine parvo virus (PPV) (which is used as a model virus for human B19parvovirus), in the purified butyrylcholinesterase, the eluate from thesecond PAM affinity chromatography step was pasteurized at about +60° C.for about 24 hours in a solution of sucrose and sodium citrate or lysineand sodium citrate or glycine and sucrose.

We found that combinations of sucrose (about 0.3 to about 0.6 M) withvarying concentrations of sodium citrate (about 0.6 to about 1.0 M) orthe combination of lysine (about 0.1 to about 0.5 M) with sodium citrate(about 1.0 M) allowed pasteurization of butyrylcholinesterase in liquidform at about +60° C. with greater than 95% butyrylcholinesteraseactivity remaining and greater than a 10⁴ reduction in PPV. The finalbutyrylcholinesterase product can be subjected to diafiltration orultrafiltration before formulation. Butyrylcholinesterase can beformulated in liquid form in about 0.1 to about 0.15 M NaCl and about0.02 M sodium phosphate buffer (pH about 6.5 to about 7.5) and stored inliquid for or it can be lyophilized.

The process may readily be scaled up. For example, when processing about150 kg of biological fluid, such as Cohn Fraction IV-1 or IV-4, werecommend using approximately 55-70 L of cation exchange resin andapproximately 7.5-10 L of PAM resin.

EXAMPLE II Affinity Purification of Butyrylcholinesterase Using PeptideLigands

Identification of positive peptides: Solid phase combinatorial peptidelibraries were synthesized on polymethacrylate beads (Buettner et al,Int. J. Pep Prot. Res., 47, 70-83 (1996)) and screened for the bindingof butyrylcholinesterase using the radiolabeled technique of Jentoft etal. (Methods in Enzymology, 91:570-79 (1983)) to radiolabelbutyrylcholinesterase. To detect positive ligands that bindbutyrylcholinesterase, the screening method of Mondorf et al. (J.Peptide Res., 52(6):526-36 (1998)) combined with an activity assay usingthe substrate butyrylthiocholine and DTNB (5,5-Dithiobis-2-NitrobenzoicAcid) were used. This stained the beads that bound activebutyrylcholinesterase yellow.

We have identified the following peptides as preferred peptide affinityligands for the concentration, separation, and purification ofbutyrylcholinesterase: AKDQIP (SEQ ID NO: 1) (alanine, lysine, asparticacid, glutamine, isoleucine, proline), AKGDQN (SEQ ID NO: 2) (alanine,lysine, glycine, aspartic acid, glutamine, asparagine), WKDAVQ (SEQ IDNO: 3) (tryptophan, lysine, aspartic acid, alanine, valine, glutamine),GFVGXA (SEQ ID NO: 4) (glycine, phenylalanine, valine, glycine, X,alanine, wherein X is 2-naphthylalanine), GFHGXI (SEQ ID NO: 5)(glycine, phenylalanine, histidine, glycine, X, isoleucine, wherein X is2-naphthylalanine), AFTNGE (SEQ ID NO: 6) (alanine, phenylalanine,threonine, asparagine, glycine, glutamic acid), AFTNQE (SEQ ID NO: 7)(alanine, phenylalanine, threonine, asparagine, glutamine, glutamicacid), GTNYHQ (SEQ ID NO: 8) (glycine, threonine, asparagine, tyrosine,histidine, glutamine), AEVDPG (SEQ ID NO: 9) (alanine, glutamic acid,valine, aspartic acid, proline, glycine).

Any of those ligands, or other butyrylcholinesterase-binding peptides,can be immobilized on an affinity matrix material, and used in affinityseparations methods to concentrate, isolate, and recoverbutyrylcholinesterase from, e.g., plasma, Cohn Fract IV-1, Cohn FractionIV-4, and other biological fluids containing butyrylcholinesterase incombination with other proteins and extraneous biological matter. Itwill be appreciated by those skilled in the art that, in view of thepresent disclosure, one of skill in the art will be able to identifyadditional such peptides having the desired level of affinity, andreversible binding properties with butyrylcholinesterase. Accordingly,the foregoing peptide ligands are illustrative only and the recitationof those ligands herein should not be construed as a limitation on thescope of the invention or of the potential ligands useful in the presentinvention.

Numerous modifications and variations of the methods of the presentinvention are possible in light of the foregoing disclosure. Therefore,one of skill in the art will understand that the invention can beexploited otherwise than as specifically described herein.

All references cited herein are incorporated herein by reference intheir entirety.

1. A method for producing purified, virally inactivatedbutyrylcholinesterase comprising: a. contacting abutyrylcholinesterase-containing biological fluid with acarboxymethyl-agarose cation exchange chromatography material; b.concentrating the fall through from the cation exchange chromatographystep by ultrafiltration; c. contacting the ultrafiltration concentratewith a procainamide affinity chromatography material to bindbutyrylcholinesterase; d. eluting butyrylcholinesterase from theprocainamide affinity chromatography material; e. solvent-detergenttreating the eluate from the procainamide affinity chromatographymaterial with about 1% (v/v) Tween-80 and about 0.3% (v/v) TnBP; f.contacting the solvent-detergent treated eluate with a procainamideaffinity chromatography material to bind butyrylcholinesterase; g.eluting butyrylcholinesterase from the procainamide affinitychromatography material; and h. pasteurizing the elutedbutyrylcholinesterase at about +60° C. for at least about 8 hours. 2.The method of claim 1, wherein the butyrylcholinesterase containingbiological fluid comprises a plasma fraction selected from the groupconsisting of Cohn Fraction IV-4, Cohn Fraction IV-1, PPT. IV from aKistler-Nitschmann fractionation, and combinations thereof.
 3. Themethod of claim 1, wherein the butyrylcholinesterase containingbiological fluid comprises recombinantly produced butyrylcholinesterase.4. A method for producing purified, virally inactivatedbutyrylcholinesterase comprising: a. contacting abutyrylcholinesterase-containing biological fluid with acarboxymethyl-agarose cation exchange chromatography material; b.solvent-detergent treating the biological fluid with about 1% (v/v)Tween-80 and about 0.3% (v/v) TnBP; c. contacting the solvent-detergenttreated fall through with a procainamide affinity chromatographymaterial to bind butyrylcholinesterase; d. recoveringbutyrylcholinesterase from the procainamide affinity chromatographymaterial; and e. pasteurizing the recovered butyrylcholinesterase atabout +60° C. for at least about 8 hours.
 5. The method of claim 4,wherein the butyrylcholinesterase containing biological fluid comprisesa plasma fraction selected from the group consisting of Cohn FractionIV-4, Cohn Fraction IV-1, PPT. IV from a Kistler-Nitschmannfractionation, and combinations thereof.
 6. The method of claim 4,wherein the butyrylcholinesterase-containing biological fluid comprisesrecombinantly produced butyrylcholinesterase.
 7. A method for producingpurified, virally inactivated butyrylcholinesterase comprising: a.contacting a butyrylcholinesterase-containing biological fluid with acation exchange chromatography material; b. subjecting the biologicalfluid to affinity purification using a peptide ligand affinitychromatography material that binds butyrylcholinesterase; c. recoveringbutyrylcholinesterase from the peptide ligand affinity chromatographymaterial; d. solvent-detergent treating the biological fluid; e.contacting the solvent-detergent treated biological fluid with a peptideligand affinity chromatography material; f. recoveringbutyrylcholinesterase from the affinity chromatography material; and g.pasteurizing the recovered butyrylcholinesterase.
 8. The method of claim7, wherein said butyrylcholinesterase containing biological fluidcomprises a plasma fraction selected from the group consisting of CohnFraction IV-4, Cohn Fraction IV-1, PPT. IV from a Kistler-Nitschmannfractionation, and combinations thereof.
 9. The method of claim 7,wherein said butyrylcholinesterase-containing biological fluid comprisesrecombinantly produced butyrylcholinesterase.
 10. The method of claim 7,wherein said pasteurized butyrylcholinesterase is formulated in about0.1 to about 0.15 M NaCl and about 0.02 M sodium phosphate.
 11. Themethod of claim 7, wherein said peptide ligand is selected from thegroup consisting of: a. alanine, lysine, aspartic acid, glutamine,isoleucine, and proline; b. alanine, lysine, glycine, aspartic acid,glutamine, and asparagine; c. tryptophan, lysine, aspartic acid,alanine, valine, and glutamine; d. glycine, phenylalanine, valine,glycine, 2-naphthylalanine, and alanine; e. glycine, phenylalanine,histidine, glycine, 2-naphthylalanine, and isoleucine; f. alanine,phenylalanine, threonine, asparagine, glycine, and glutamic acid; g.alanine, phenylalanine, threonine, asparagine, glutamine, and glutamicacid; h. glycine, threonine, asparagine, tyrosine, histidine, glutamine;and i. alanine, glutamic acid, valine, aspartic acid, proline, glycine.12. A method for producing purified, virally inactivatedbutyrylcholinesterase comprising: a. contacting abutyrylcholinesterase-containing biological fluid with a cation exchangechromatography material; b. solvent-detergent treating the biologicalfluid; c. contacting the biological fluid with at least one peptideligand affinity chromatography material that bindsbutyrylcholinesterase; d. recovering butyrylcholinesterase from thepeptide ligand affinity chromatography material; and e. pasteurizing theeluted butyrylcholinesterase.
 13. The method of claim 12, wherein saidbutyrylcholinesterase containing biological fluid comprises a plasmafraction selected from the group consisting of Cohn Fraction IV-4, CohnFraction IV-1, PPT. IV from a Kistler-Nitschmann fractionation, andcombinations thereof.
 14. The method of claim 12, wherein saidbutyrylcholinesterase-containing biological fluid comprisesrecombinantly produced butyrylcholinesterase.
 15. The method of claim12, wherein said pasteurized butyrylcholinesterase is formulated inabout 0.1 to about 0.15 M NaCl and about 0.02 M sodium phosphate. 16.The method of claim 12, wherein said at least one peptide ligand is apeptide selected from the group consisting of: a. alanine, lysine,aspartic acid, glutamine, isoleucine, and proline; b. alanine, lysine,glycine, aspartic acid, glutamine, and asparagine; c. tryptophan,lysine, aspartic acid, alanine, valine, and glutamine; d. glycine,phenylalanine, valine, glycine, 2-naphthylalanine, and alanine; e.glycine, phenylalanine, histidine, glycine, 2-naphthylalanine, andisoleucine; f. alanine, phenylalanine, threonine, asparagine, glycine,and glutamic acid; g. alanine, phenylalanine, threonine, asparagine,glutamine, and glutamic acid; h. glycine, threonine, asparagine,tyrosine, histidine, glutamine; and i. alanine, glutamic acid, valine,aspartic acid, proline, glycine.
 17. A method for isolatingbutyrylcholinesterase comprising contacting abutyrylcholinesterase-containing biological fluid with a peptide ligandaffinity chromatography material wherein the peptide ligand is selectedfrom the group consisting of: a. alanine, lysine, aspartic acid,glutamine, isoleucine, and proline; b. alanine, lysine, glycine,aspartic acid, glutamine, and asparagine; c. tryptophan, lysine,aspartic acid, alanine, valine, and glutamine; d. glycine,phenylalanine, valine, glycine, 2-naphthylalanine, and alanine; e.glycine, phenylalanine, histidine, glycine, 2-naphthylalanine, andisoleucine; f. alanine, phenylalanine, threonine, asparagine, glycine,and glutamic acid; g. alanine, phenylalanine, threonine, asparagine,glutamine, and glutamic acid; h. glycine, threonine, asparagine,tyrosine, histidine, glutamine; and i. alanine, glutamic acid, valine,aspartic acid, proline, glycine; and recovering butyrylcholinesterasebound to said peptide ligand chromatography material.
 18. The method ofclaim 17, wherein the peptide ligand is covalently bound to a supportselected from the group consisting of agarose, dextran, cellulose,polyacrylamide, polystyrene, acrylic polymers, vinyl polymers, silica,and cross-linked versions thereof.
 19. The method of claim 17, furthercomprising contacting the biological fluid with a cation exchangechromatography material.
 20. The method of claim 17, further comprisingsubjecting the biological fluid to a solvent-detergent treatment step.21. The method of claim 17, further comprising subjecting the biologicalfluid to a pasteurization step.